1. Field of the Invention
The present invention relates to novel compounds which are xcex2-lactamase inhibitors, pharmaceutical compositions containing the same and methods of inhibiting xcex2-lactamases. More specifically this invention is concerned with novel 7-alkylidene cephalosporins and pharmaceutically acceptable salts thereof.
2. Description of the Background
The most important mechanism of microbial resistance to xcex2-lactam antibiotics is the bacterial production of xcex2-lactamases, enzymes which hydrolytically destroy xcex2-lactan antibiotics, such as penicillins and cephalosporins. This type of resistance can be transferred horizontally by plasmids that are capable of rapidly spreading the resistance, not only to other members of the same strain, but even to other species. Due to such rapid gene transfer, a patient can become infected with different organisms, each possessing the same xcex2-lactamase.
xcex2-lactamase enzymes have been organized into four molecular classes: A, B, C, and D based on amino acid sequence. Class A, which includes RTEM and the xcex2-lactamase of staphylococcus aureus, class C, which includes the lactamase derived from P99 Enterobacter cloacae, and class D are serine hydrolases. Class A enzymes have a molecular weight of about 29 kDa and preferentially hydrolyze penicillins. The class B lactamases are metalloenzymes and have a broader substrate profile than the proteins in the other classes. Class C enzymes include the chromosomal cephalosporinases of gram-negative bacteria and have molecular weights of approximately 39 kDa. The recently recognized class D enzymes exhibit a unique substrate profile which differs significantly from both class A and class C.
The class C cephalosporinases, in particular, are responsible for the resistance of gram-negative bacteria to a variety of both traditional and newly designed antibiotics. The Enterobacter species, which possess a class C enzyme, are now the third greatest cause of hospital-acquired infections in the United States. This class of enzymes often has poor affinities for inhibitors of the class A enzymes, such as clavulanic acid, a commonly prescribed inhibitor, and to common in vitro inactivators, such as 6-xcex2-iodopenicillanate.
One strategy for overcoming rapidly evolving bacterial resistance is the synthesis and administration of xcex2-lactamase inhibitors. Frequently, xcex2-lactamase inhibitors do not possess antibiotic activity themselves and are thus administered together with an antibiotic. One example of such a synergistic mixture is xe2x80x9caugmentinxe2x80x9d, which contains the antibiotic amoxicillin and the xcex2-lactamase inhibitor, clavulanic acid.
It is thus desirable to find novel xcex2-lactamase inhibitors which can be coadministered with a xcex2-lactam antibiotic.
Accordingly, one object of the present invention is to provide novel xcex2-lactamase inhibitors.
It is another object of the present invention to provide pharmaceutical compositions useful for inhibiting a xcex2-lactamase.
It is another object of the present invention to provide pharmaceutical compositions with increased xcex2-lactam antibiotic activity.
It is another object of the present invention to provide methods of inhibiting a xcex2-lactamase.
It is another object of the present invention to provide methods of enhancing the biological activity of a xcex2-lactam antibiotic.
These and other objects, which will become apparent during the following detailed description, have been achieved by the inventors"" discovery that compounds of the formula (1) 
wherein n is 0 or 1 (the sulfide or the sulfone, respectively);
R1 and R2 are the same or different and are selected from the group consisting of
a) hydrogen;
b) linear or branched C1-10-alkyl;
c) halogen;
d) hydroxy-C1-10-alkyl;
e) C1-10-alkoxy;
f) C2-10-alkanoyloxy;
g) C2-10-alkene;
h) C2-10-alkene substituted with one or more groups selected from the group consisting of chlorine, fluorine, bromine or phenyl;
i) C 1-10-alkoxycarbonyl;
j) C1-10-alkoxycarbamido;
k) Nxe2x80x94C1-10-alkoxy-Nxe2x80x94C1-10-alkylaminocarbonyl;
l) halo-C1-10-alkyl;
m) C6-10-aryl;
n) C6-10-aryl substituted with one or more groups selected from the group consisting of ethyl, n-propyl, isopropyl, amino, methylamino and dimethylamino;
o) a C2-10-heterocycle having from 1-3 heteroatoms selected from the group consisting of O, N and S; and,
p) xe2x80x94COOH or xe2x80x94COOY, wherein Y is pharmaceutically acceptable cation;
R3 is selected from the group consisting of
1) xe2x80x94COOH;
2) chlorine or fluorine;
3) trifluoromethyl;
4) xe2x80x94CHO; and,
5) xe2x80x94CH2M where M is selected from the group consisting of
a) hydrogen;
b) halogen;
c) hydroxy;
d) C1-10-alkoxy;
e) C6-10-aryloxy;
f) C6-10-aryl-C1-10-alkoxy;
g) mercapto;
h) mercapto substituted with one or more groups selected from the group consisting of methyl, ethyl or phenyl;
i) C2-10-acylthio;
j) C2-10-acyloxy or carbamoyloxy;
k) C2-10-acyloxy or carbamoyloxy substituted with one or more groups selected from the group consisting of xe2x80x94COOH, aminophenyl, phenyl, C1-6alkyl, chlorine, bromine or fluorine;
l) a quaternary ammonium salt;
m) amino or amido; and,
n) amino or amido substituted with one or more groups selected from the group consisting of C1-10-alkyl groups;
R4 is selected from the group consisting of
a) hydrogen; and,
b) pharmaceutically acceptable cations; are effective xcex2-lactamase inhibitors.
Thus, in a first embodiment, the present invention provides novel compounds of the formula (1) 
wherein n is 0 or 1;
R1 and R2 are the same or different and are selected from the group consisting of
a) hydrogen;
b) linear or branched C1-10-alkyl, preferably, C1-6-alkyl, more preferably, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, cyclopropyl, cyclopentyl, or cyclohexyl, most preferably t-butyl;
c) halogen, preferably Br or Cl;
d) hydroxy-C1-10,-alkyl, preferably, hydroxy-C1-6-alkyl, more preferably, hydroxymethyl, 1-hydroxyethyl or 2-hydroxyethyl;
e) C1-10-alkoxy, preferably, C1-6-alkoxy, more preferably, t-butoxy or methoxy;
f) C2-10-alkanoyloxy, preferably, C2-6-alkanoyloxy, more preferably, acetoxy or propanoyloxy;
g) C2-10-alkene, preferably, C2-6-alkene, more preferably, ethylene, 1-propylene or 2-propylene;
h) substituted C2-10-alkene, preferably, C2-6-alkene, more preferably ethylene, 1-propylene or 2-propylene, wherein said substituents are one more groups selected from the group consisting of chlorine, fluorine, bromine or phenyl;
i) C1-10-alkoxycarbonyl, preferably, C1-6-alkoxycarbonyl, more preferably, methoxycarbonyl or t-butoxycarbonyl;
j) C1-10-alkoxycarbamido, preferably, C1-6-alkoxycarbamido, more preferably, methoxycarbamido, ethoxycarbamido or n-propoxycarbamido
k) Nxe2x80x94C1-10-alkoxy-Nxe2x80x94C1-10-alkylaminocarbonyl, preferably, N-C1-6-alkoxy-Nxe2x80x94C1-6-alkylaminocarbonyl, more preferably, N-methoxy-N-methylaminocarbonyl, N-ethoxy-N-methylaminocarbonyl, N-methoxy-N-ethylaminocarbonyl or N-ethoxy-N-ethylaminocarbonyl;
l) halo-C1-10-alkyl, preferably, halo-C1-6-alkyl, more preferably, chloromethyl, 1-chloroethyl or 2-chloroethyl;
m) C6-10-aryl group, preferably, phenyl, tolyl, anisoyl, mesityl, and xylyl;
n) substituted C1-10-alkyl, preferably, phenyl, tolyl, anisoyl, mesityl, and xylyl, wherein said substituents are one or more groups selected from the group consisting of ethyl, n-propyl, isopropyl, amino, methylamino and dimethylamino;
o) a C2-10-heterocycle having from 1-3 heteratoms selected from the group consisting of O, N and S, preferably, triazolyl, triazinyl, oxazoyl, isoxazolyl, oxazolidinoyl, isoxazolidinoyl, thiazolyl, isothiazoyl, pyrazolyl, imidazolyl, pyrrolyl, pyrazinyl, pyridinyl, morpholinyl, quinolinyl, isoquinolinyl, indolyl, and pyrimidinyl, more preferably, pyridinyl; and,
p) xe2x80x94COOH or xe2x80x94COOY, wherein Y is a pharmaceutically acceptable cation, preferably, sodium, potassium, calcium, or any other pharmaceutically acceptable cation known in the art;
R3 is selected from the group consisting of
1) xe2x80x94COOH;
2) Cl or F;
3) trifluoromethyl;
4) xe2x80x94CHO; and,
5) xe2x80x94CH2M, wherein M is selected from the group consisting of
a) hydrogen;
b) halo, preferably F, Cl, Br, or I;
c) hydroxy;
d) C1-10-alkoxy, preferably, C6-10-alkoxy, more preferably, methoxy, ethoxy, n-propoxy or isopropoxy;
e) C6-10-aryloxy, preferably, C6-10-aryloxy, more preferably, phenoxy or naphthoxy;
f) C6-10-aryl-C1-10-alkoxy, preferably, C6-10-aryl-C1-6-alkoxy, more preferably, phenylmethoxy, 1-phenylethoxy or 2-phenylethoxy;
g) mercapto, preferably, thiol;
h) substituted mercapto, preferably, thiol, wherein said substituents are selected from the group consisting of methyl, ethyl or phenyl;
i) C2-10-acylthio, preferably C2-6-acylthio, more preferably, acetylthio or propanoylthio;
j) C2-10-acyloxy or carbamoyloxy, preferably, C2-6-alkanoyloxy, C6-10-aryl-carbonyloxy, carbamoyloxy or thiocarbamoyloxy, more preferably, acetoxy or benzoyloxy;
k) substituted C2-10-acyloxy or carbamoyloxy, preferably, C2-6-alkanoyloxy, C6-10-aryl-carbonyloxy, Nxe2x80x94C1-6-alkylcarbamoyloxy, N,N-di-C1-6-alkylcarbamoyloxy, thiocarbamoyloxy, Nxe2x80x94C1-6-alkylthiocarbamoyloxy or N,N-di-C1-6-alkylthiocarbamoyloxy, more preferably, acetoxy, xcex1-aminophenylacetoxy, benzoyloxy, benzyloxycarbonyloxy, succinoyloxy, N-methylcarbamoyloxy, N-ethylcarbamoyloxy, N,N-dimethylcarbamoyloxy, N,N-diethylcarbamoyloxy, N-methylthiocarbamoyloxy, N-ethylthiocarbamoyloxy, N,N-dimethylthiocarbamoyloxy, N,N-diethylthiocarbamoyloxy, wherein said substituents are one or more groups selected from the group consisting of xe2x80x94COOH, aminophenyl, phenyl, methyl, ethyl, chlorine, bromine or fluorine;
l) a quaternary ammonium salt, preferably trimethyl ammonium chloride or triethyl ammonium chloride;
m) amino or amido group, preferably xe2x80x94NH2 or xe2x80x94CONH2; and,
n) substituted amino or amido group, preferably xe2x80x94NH2 or xe2x80x94CONH2, wherein said substituents are one or two C1-10-alkyl groups, preferably C1-6-alkyl groups, more preferably, methyl, ethyl, n-propyl, isopropyl or n-butyl;
R4 is selected from the group consisting of
a) hydrogen; and,
b) pharmaceutically acceptable cations, preferably, sodium, potassium or calcium.
In a preferred embodiment, n is 0, R2 is hydrogen and R1 is selected from the group consisting of t-butyl, phenyl, pyridyl, COMe and N-methyl-N-methoxy-aminocarbonyl.
In a more preferred embodiment, n is 0, R2 is hydrogen, and R1 is selected from the group consisting of Co2-t-Bu and CHO.
In another preferred embodiment, n is 1, R2 is hydrogen, and R1 is selected from the group consisting of CO2Me, CH2OH and t-butyl.
In another more preferred embodiment, n is 1, R2 is hydrogen and R1 is selected from the group consisting of phenyl, pyridyl and CO2-t-butyl.
In another preferred embodiment, n is 0 and R1 and R2 are the same and are selected from the group consisting of bromine and chlorine.
In another preferred embodiment, n is 1 and R1 and R2 are the same and are selected from the group consisting of bromine and chlorine.
In another preferred embodiment, n is 0, R1 is hydrogen, and R2 is bromine.
In another preferred embodiment, n is 1, R1 is hydrogen, and R2 is selected from the group consisting of phenyl and bromine.
In a most preferred embodiment, n is 1, R1 is pyridyl, R2 is hydrogen, R3 is xe2x80x94CH2OAc and R4 is sodium.
Compounds according to formula (1) were obtained as follows. 7-Aminocephalosporanic acid (commercially available from Aldrich), esterified with diphenyl diazomethane (2), was treated with excess triethylamine and trifluoromethanesulfonic anhydride and the resultant trifluorosulfonyl imine was hydrolyzed to produce benzhydryl 7-oxocephalosporanate 3. (See Hagiwara, D. F.; Sawada, K.; Ohnami, T.; Aratani, M.; Hashimoto, M. J. Chem Soc. Chem. Commun. 1982, 578.) Due to its instability, 3 was used directly in the next step without purification.
The 7-alkylidenecephalosporanates 4 were prepared by treating 7-oxocephalosporanate 3 with the corresponding Wittig reagent at xe2x88x9278xc2x0 C. Compounds 4 a-k were prepared in the standard manner with the exceptions of 4b, 4j, 4l and 4m. Compound 4b required the addition of the Zn/Cu couple to 3 in the presence of CCl4 and PPh3 to lead to its formation. Compound 4j was prepared by the reduction of 4i with NaCNBH3. Compound 4a was reduced by the Zn/Cu couple to produce monobromomethylenecephem 4l, which was further treated with t-BULi and CUCN to give compound 4m as shown below.
Many of the compounds in the series 4 were oxidized with excess m-CPBA yielding the corresponding sulfones 5. Deprotection of compounds 4 and 5 gave the corresponding sodium salts 6 and 7 as shown below.
Compounds containing R1 and R2 groups (i.e., alkoxy or alkene) not shown above may be synthesized by using an appropriate Wittig reagent R1R2Cxe2x95x90PP3. The Wittig reagents ROCHxe2x95x90PPh3 and H2Cxe2x95x90CHxe2x80x94CHxe2x95x90PPh3 may be used to make the 7-alkyoxymethylene and 7-alkenylmethylene compounds, respectively.
In addition to the Wittig reaction, the Peterson olefination procedure may be used to form 7-substituted methylene compounds from the oxocephalosporanate 3. For example, (RO) (SiMe3)CHLi or (haloalkyl)(SiMe3)CHLi may add to 3 to form the 7-alkoxymethylene or 7-halomethylmethylene compounds, respectively.
The 7-alkanoylmethylene species may be made by forming the vinyl anion and reacting it with a desirable alkanoyl halide. The vinyl anion may be made by a standard lithium-halogen (or magnesium-halogen) exchange reaction, for example, reaction of 4a with methyl lithium. The lithium vinyl group may then be functionalized by reaction with an an alkoxycarbonyl chloride.
The 7-carboxylmethylene compounds (R1 or R2xe2x95x90COOH or COOY) may be formed by hydrolysis of the corresponding ester, preferably, the corresponding t-butyl ester.
The compounds wherein R3 is a halogen may be formed by displacement of the xe2x80x94OAc group with ethylxanthate (EtOCS2K). Raney-Nickel desulfurization (H2/Raxe2x80x94Ni) would yield the exocyclic alkene which may then be ozonized to the 3-hydroxy cephem. Reaction with a halogenating reagent would provide the 3-halo species. For example, PCls may be used to convert the 3-OH group into a 3-Cl group. The 3-methyl species may be obtained by the rearrangement of the exocyclic alkene, formed by Raney-Nickel desulfurization, by reaction with Et3N. The 3-hydroxymethyl species may be obtained by hydrolysis of the xe2x80x94OAc group with NaOH or an appropriate enzyme. The 3-halomethyl species may be formed by reaction of the 3-hydroxymethyl species with a halogenating reagent. For example, PCl5 may be used to form the 3-chloromethyl species.
The compounds wherein M is alkoxy, aryloxy, or arylalkoxy may be obtained by reaction of the 3-hydroxymethyl species with tosyl chloride and displacement of the resultant tosylate with an oxide. For example, sodium methoxide may be used to obtain the 3-methoxymethyl species. The compounds wherein M is mercapto may be formed by reaction of the 3-chloromethyl compound with sodium sulfhydride (NaSH). This compound may further be derivatized with an alkylhalide to form a substituted mercapto or an acylchloride to form an acylthio group.
The species wherein M is an amino group may be formed by the Gabriel Synthesis, i.e., reaction of the 3-chloromethyl compound with potassium phthalimide and hydolysis of the product with acid to yield the 3-aminomethyl compound. The 3-ammoniomethyl compound may be formed by reaction of the 3-aminomethyl compound with methyl chloride to form the 3-trimethylammoniomethyl chloride.
The compound wherein N is an amido group (CONH2) may be formed by displacement of the tosylate described above with cyanide, e.g., KCN, followed by hydrolysis of the resulting nitrile to the amide.
The aforementioned salts of 7-alkylidene cephems were evaluated as inhibitors of the Class C xcex2-lactamase of Enterobacter cloacae P99 and TEM2 by relative IC50 analysis. The IC50 value represents the concentration of inhibitor required to effect a 50% loss of activity of free enzyme. The IC50 value of each compound was determined as follows. Following a 10 minute incubation of a dilute solution of enzyme (2.56 nm) and inhibitor ( less than 0.64 mM), a 50 mL aliquot of this incubation mixture was then further diluted into 1 mL nitrocefin solution, and the rate of hydrolysis was measured during a 1 minute period by monitoring the absorbance of nitrocefin as a function of time. In addition, the IC50 values of tazobactam and clavulanic acid were determined as relative controls. The data is presented in Table 1 below.
Compound 7e, 7-(Z)-[(2-pyridyl)methylene] cephalosporanic acid sulfone, was determined to be more potent than tazobactam, showing a 20 fold increase in activity. In general, it was found that the sulfones were more potent than their corresponding sulfide analogs. One striking example of this is the 1300 fold increase in activity of 7e, the pyridyl sulfone, over it""s sulfide 6e.
In a second embodiment, the present invention provides pharmaceutical compositions useful for inhibiting a xcex2-lactamase. The present pharmaceutical compositions comprise at least one of the present 7-vinylidene cephalosporins and at least one pharmaceutically acceptable carrier.
The present compositions may be employed in capsule form or as tablets, powders or liquid solutions or as suspensions or elixirs. They may be administered orally, intravenously or intramuscularly. The present compositions are preferably presented in a form suitable for absorption by the gastro-intestinal tract.
Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional pharmaceutical carriers such as binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example, lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine, lubricants, for example, magnesium stearate, talc, polyethylene glycol, silica; disintegrants, for example, potato starch; or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in the art. Oral liquid preparations may be in the form of aqueous or oily suspensions, solutions, emulsions, syrups, elixirs, etc. or may be presented as a dry product, for reconstruction with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example, sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel or hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate or acacia; non-aqueous vehicles, which may include edible oils, for example, almond oil, fractionated coconut oil, oily esters, propylene glycol, or ethyl alcohol; preservatives, for example, methyl or propyl p-hydroxybenzoates or sorbic acid. Suppositories will contain conventional suppository bases, e.g., cocoa butter or other glyceride.
Compositions for injection may be presented in unit does form in ampules, or in multidose containers with an added preservative. The compositions may take such forms as suspensions, solutions, emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
The present compositions may also be prepared in suitable forms for absorption through the mucous membranes of the nose and throat or bronchial tissues and may conveniently take the form of powder or liquid sprays or inhalants, lozenges, throat paints, etc. For medication of the eyes or ears, the preparations may be presented as individual capsules, in liquid or semi-solid form, or may be used as drops, etc. Topical applications may be formulated in hydrophobic or hydrophilic bases as ointments, creams, lotions, paints, powders, etc.
Also, in addition to a carrier, the present compositions may include other ingredients such as stabilizers, binders, antioxidants, preservatives, lubricators, suspending agents, viscosity agents or flavoring agents and the like.
For veterinary medicine, the composition may, for example, be formulated as an intramammary preparation in either long acting or quick-release bases.
The dosage to be administered depends to a large extent upon the condition of the subject being treated and the weight of the subject, the route and frequency of administration, the parenteral route being preferred for generalized infections and the oral route for intestinal infections.
The instant compositions may be administered in several unit dosage forms as, for example, in solid or liquid orally ingestible dosage form. The compositions per unit dosage, whether liquid or solid may contain from 0.1% to 99% of active material (the present 7-vinylidene cephalosporins and optional antibiotic), the preferred range being from about 10-60%. The composition will generally contain from about 15 mg to about 1500 mg by weight of active ingredient based upon the total weight of the composition; however, in general, it is preferable to employ a dosage amount in the range of from about 250 mg to 1000 mg. In parenteral administration the unit dosage is usually the pure compound in a slightly acidified sterile water solution or in the form of a soluble powder intended for solution.
The present xcex2-lactamase inhibitors will be particularly useful in the treatment of infections caused by Enterobacter, Citrobacter, and serratia. These bacteria have the ability to attach to the epithelial cells of the bladder or kidney (causing urinary tract infections) and are resistant to multiple antibiotics including amoxicillin and ampicillin. The present xcex2-lactamase inhibitors would also be useful in the treatment of infections caused by highly resistant Pneumococci. Such diseases include otitis media, sinusitis, meningitis (both in children and adults), bacteremia, and septic arthritis. Resistant pneumococcal strains have surfaced in many parts of the world. For example, in Hungary, 58% of S. pneuzmoniae are resistant to penicillin, and 70% of children who are colonized with S. pneumoniae carry resistant strains that are also resistant to tetracycline, erythromycin, trimethoprin/sulfamethoxazole (TMP/SMX), and 30% resistant to chloroanphenicol. Klebsiella pneumoniae (resistant via the production of xcex2-lactamase) have caused hospital outbreaks of wound infection and septicemia.
Thus, in a third embodiment, the present invention provides pharmaceutical compositions with increased xcex2-lactam antibiotic activity. This pharmaceutical composition is as defined above, but in addition to at least one of the present 7-vinylidene cephalosporins and at least one a pharmaceutically acceptable carrier, the compositions also contains at least one xcex2-lactam antibiotic. The xcex2-lactam antibiotic may be any of the above-noted antibiotics or any other known in the art, preferably amoxicillin or piperacillin, and its selection will depend upon what indication is necessary.
In a fourth embodiment, the present invention provides a method of inhibiting a xcex2-lactamase, comprising administering to a patient in need thereof an effective amount of at least one of the present 7-vinylidene cephalosporins. The method of administration may be any of the above-noted methods or any other Known to one of skill in the art.
In a fifth embodiment, the present invention provides a method of enhancing the biological activity of a xcex2-lactam antibiotic by coadministering to a patient in need thereof, an effective amount of one of the present 7-vinylidene cephalosporins and an effective amount of at least one xcex2-lactam antibiotic. The method of administration may be any of the above-noted methods or any other known to one of skill in the art. The xcex2-lactam antibiotic may be any of the above-noted xcex2-lactam antibiotics or any other known in the art.
Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof.